This invention relates generally to the field of genetic engineering in plants. This invention relates more specifically to methods of enhancing traits in plants, methods of determining plant growth regulator signal transduction pathways, and methods of utilizing plant flavin-containing monooxygenases.
Auxin Biosynthesis and Effects
Auxin is an essential plant hormone that influences many aspects of plant growth and development including cell division and elongation, differentiation, tropisms, apical dominance, senescence, abscission and flowering. (Hooley, Plant Cell (1998) 10:1581-4). Not only is auxin a plant growth regulator, it is also likely to be a morphogen. (Sabatini et al., Cell (1999) 99:463-472). Although auxin has been studied for more than 100 years, its biosynthesis, transport, and signaling pathways remain elusive. In order to understand the biological functions of auxin, it is necessary to elucidate how auxin is synthesized, transported, and used as a signaling agent.
Indole-3-acetic acid (IAA), the first auxin to be chemically identified, appears to be the major endogenous auxin. (Davies, The Plant Hormones: Their Nature, Occurrence, and Functions (1995) Kluwer Academic Publishers, 1-12). Based on its structural similarities, tryptophan has been proposed as the auxin biosynthesis precursor. (Bartel, Ann Rev Plant Physiol (1997) 48:51-66). Many pathways have been proposed for converting tryptophan to IAA, but at present, none has been definitely proven. Tryptophan can be converted to indole-pyruvate by transferring the amino group. Indole-pyruvate can be further converted to indole-acetaldehyde, which can be oxidized to IAA. Tryptamine, a decarboxylated product of tryptophan, has also been proposed as an auxin biosynthesis intermediate. It was proposed that tryptamine is converted to indole-acetaldehyde by amine oxidase.
In addition, a P450-type monooxygenase has recently been found to catalyze the conversion of tryptophan to indole-acetaldoxime that can then be converted to indole-acetonitrile. (Bartel, Ann Rev Plant Physiol (1997) 48:51-66). Acetonitrile can be used as nitrilase substrate to generate auxin. In bacteria, tryptophan is converted to indole-acetamide by tryptophan monooxygenase. In addition, experiments with tryptophan auxotroph mutants have revealed a tryptophan-independent auxin biosynthesis pathway. In light of the fact that there are many possible auxin synthesis pathways, it is not surprising that no auxin deficient mutants have been isolated in the many genetic screens that have been carried out using a variety of plant species.
Another approach in finding auxin mutants has been to isolate gain-of-function mutants, for example, auxin overproduction mutants. Several Arabidopsis mutants having elevated free auxin levels have been isolated from EMS mutagenesis screens. To date, all reported auxin overproducing mutants are recessive mutations. Mutations at the superroot locus (surl) (Boejan et al., Plant Cell (1995) 7:1405), which is allelic to rooty (King et al., Plant Cell (1995) 7: 2023), hookless3 (Lehman et al., Cell (1996) 85:183), and alf1 (Luschnig et al., Genes Dev (1998) 12:2175), produce a phenotype having lateral root proliferation, epinastic leaves, and long hypocotyls. Sur2 mutants (Delarue et al., Plant J (1998) 14:603-611) have higher endogenous free auxin levels, but mutations at the sur2 locus are neither genetically nor phenotypically stable. In homozygous sur2 populations, a few plants having the wild-type (nonmutant) phenotype are observed in each generation. Homozygous sur1 usually does not produce true leaves and never sets any seeds. Thus, although cloning of sur1 and sur2 will help in understanding the auxin biosynthetic pathway, the infertile SUR mutant loci are not the most desirable for identification of other cellular components involved in auxin biosynthesis. Instead, a fertile auxin overproducing mutant would be more useful than the sterile auxin overproducing mutants that have so far been isolated.
Auxin is believed to be synthesized in the shoot and, for example, transported to the root tip to initiate root growth and elongation. Polar auxin transport is mediated by auxin efflux carriers and influx carriers. PIN (Steinmann et al., Science (1999) 286:316; Galweiler et al., Science (1998) 282:2226) and EIR1/PIN2/AGR1 (Marchant et al., Embo J (1999) 18:2066; Muller et al., Embo J (1998) 17:6903), which are homologous to bacterial membrane transporters, are considered to be putative efflux carriers in the shoot and root respectively. On the other hand, AUXI (Marchant et al., Embo J (1999) 18:2066), an amino acid permease homolog, is considered a possible auxin influx carrier. Other components including TIR3 have also been found to be important for auxin polar transport. Since auxin polar transport is important for plant tropisms and plant development, understanding this process is useful for determining the molecular basis in regulating plant pattern and development. A dominant auxin overproducing mutant would be very useful in defining how auxin transport is involved in regulation of plant organ development.
Some of the mechanisms involved in auxin signaling are better understood than the pathways of auxin biosynthesis and transport. The auxin signaling components that are presently known have been identified either by biochemical methods or by genetic screens for altered response to exogenously added auxin. In one method, following auxin treatment, the AUX/IAA genes were found to be rapidly and specifically induced and to express short-lived transcriptional repressors (Abel and Theologis, Plant Physiol (1996) 111:9); however, the expression pathway is not fully understood. AUX/IAA genes were also found to associate with other DNA binding proteins such as auxin response factors (ARFs) to modulate gene expression. (Hooley, Plant Cell (1998) 10:1581). Another class of auxin response mutants showed a diminished response to exogenous auxin, including the axr1 or TIR1 mutants, which are thought to regulate protein turnover in an auxin dependent manner. (Leyser et al. Nature (1993) 364:161; Ruegger et al., Genes Dev (1998) 12:198) The axr1 gene product AXR1 is a component of the ubiquitin-mediated protein degradation pathway.
All the components of the auxin signaling pathways that have been identified so far are components that act downstream from the auxin sensing step. Therefore, gaps in knowledge remain in the current understanding of the auxin signaling pathway that begins with auxin sensing, through activation of transcription factors, to downstream effectors. A dominant auxin-overproducing mutant would provide an ideal tool for identifying components of the auxin signaling pathway.
Flavin-Containing Monooxygenases
Flavin-containing monooxygenases (FMOs) have a unique ability to oxidize structurally dissimilar compounds. Compared to the P450 family of monooxygenases, there are relatively few FMOs. Some FMOs have a plurality of isozymes, but the precise characterization of and specific function of each isozyme is not well understood.
The FMO group of enzymes is known to be important in the metabolism of a variety of drugs and toxins. The relative abundance of FMOs in most mammalian tissues and their loose substrate specificity suggests that they contribute substantially to detoxification of xenobiotics. The FMOs of rat, pig and rabbit are microsomal xenobiotic-metabolizing enzymes which oxidize various xenobiotics including drugs, agricultural chemicals, and environmental pollutants. It has recently been shown that population-specific polymorphisms of the human FMO3 gene have significance for detoxification of chemicals to protect humans from the potentially toxic properties of drugs and chemicals. (Cashman, Drug Metab Dispos (2000) 28:169).
The present invention provides methods for using enhanced expression of nucleotide sequences encoding flavin-containing monooxygenases, hereinafter xe2x80x9cFMOsxe2x80x9d, to elicit desired traits, determine biochemical pathways, and oxidize xenobiotics. In one embodiment, the flavin-containing monooxygenase is from Arabidopsis thaliana and the FMO is an Arabidopsis thaliana-FMO (AT-FMO).
Embodiments of the present invention provide a method of enhancing at least one trait in a plant comprising transforming a plant with an expression vector encoding at least one FMO, expressing the FMO(s) encoded by the vector, and measuring the trait(s). The enhanced traits include, but are not limited to, enhanced expression of a FMO, increased hypocotyl elongation, increased root thickness, increased root hair development, increased lateral root initiation, increased apical dominance, epinastic leaf growth, increased flowering node formation, increased fruit yield, increased endogenous auxin levels, parthenocarpic fruit production, altered gene expression, altered pathogen resistance, altered pest resistance, and altered herbicide resistance. In addition, the trait may convey altered sensitivity to a plant growth regulator selected from the group consisting of auxins and chemicals having auxin-like activity, gibberellins, cytokinins, abscisic acid, ethylene, brassinosteroids, salicylates, and jasmonates.
Embodiments of the present invention provide a plant transformed with an expression vector encoding at least one FMO, wherein one or more sequences encoding FMO(s) are operably linked with one or more promoters or other regulatory sequences. Embodiments of the present invention further provide a plant transformed with an expression vector encoding at least one FMO, wherein expression of the FMO(s) may be regulated by a constitutive promoter, an inducible promoter, a tissue-specific promoter, or other promoters suitable for use in the invention.
Embodiments of the present invention further provide a method for identifying steps of auxin biosynthesis by transforming a plant with an expression vector encoding at least one FMO, expressing the FMO(s) encoded by the vector, carrying out a suppression screening of transformed plant material expressing the FMO(s), and measuring auxin biosynthesis. In other embodiments of the invention, suppression screening to identify steps of auxin biosynthesis include, but are not limited to, use of screening techniques wherein loss-of-function mutations are exposed, and screening techniques wherein gain-of-function mutations are exposed.
Another embodiment of the present invention provides a method for identifying steps of signal transduction pathways by transforming a plant with an expression vector encoding at least one FMO, expressing the FMO(s), carrying out suppression screening of transformed plants expressing the FMO(s), and measuring altered signal transduction. In other embodiments of the invention, suppression screening to identify steps of signal transduction pathways include, but are not limited to, use of screening techniques wherein loss-of-function mutations are exposed, and screening techniques wherein gain-of-function mutations are exposed.
Another embodiment of the present invention further provides a method for identifying a plant growth regulator by exposing a substrate molecule to at least one FMO to generate a modified substrate molecule, and measuring plant responses to the modified substrate molecule. In one embodiment, more than one modified substrate molecule is produced, and responses to more than one substrate molecule are measured. In another embodiment, responses to one or more modified substrate molecule are measured using a gene array. In yet another embodiment, responses to one or more modified substrate molecule are measured by measuring traits including, but not limited to, auxin levels, hypocotyl length, hypocotyl cell length, cotyledon thickness, cotyledon length, lateral root initiation, root hair number, leaf thickness, leaf epinasty, leaf angle, number of flowering nodes, flowering phenology, fruit development, parthenocarpy, protein content, amino acid profile, gene transcription, and gene expression.
Yet another embodiment of the invention further provides a method for studying the interaction between at least two growth regulators by transforming a plant with an expression vector encoding at least one FMO, expressing the FMO(s), and measuring responses to growth regulators. In one embodiment, at least one plant growth regulator is externally applied.
Still another embodiment of the invention is a method for studying root development comprising transforming a plant with an expression vector encoding at least one FMO, expressing the FMO(s), and measuring indicia of root development including, but not limited to, length, diameter, morphology, rate of elongation, root hair development, lateral root initiation, anthocyanin content, and root orientation. One embodiment provides a method for studying root development in which the plant transformed with, and expressing, at least one FMO is treated with an auxin transport inhibitor and development of club root morphology is measured.
Another embodiment of the invention provides a method for enhancing tryptamine degradation in plants transformed with and expressing at least one FMO.
One embodiment of the present invention provides a method for identifying at least one xenobiotic compound in a sample, where the xenobiotic is exposed to at least one FMO and the oxidized xenobiotic is measured by means that include, but are not limited to, chromatographic analysis, mass spectrometry, or biological assays. This method for identifying at least one xenobiotic can be used as a drug screening assay.
The FMO(s) can be used to detoxify a xenobiotic. In addition, a method is provided for producing an enzyme having xenobiotic detoxifying activity by transforming a host cell with an expression vector encoding at least one FMO and recovering fractions containing FMO activity.
Embodiments of the present invention further provide a light-sensitive promoter useful for placing downstream nucleotide sequences under regulation by specific wavelengths of light.
Another embodiment of the present invention provides a method of identifying inhibitors of FMO(s) by exposing FMO(s), or plants expressing FMO(s) to potential inhibitors and measuring FMO activity.
The above and other embodiments, features, and advantages of the present invention will be better understood from the following detailed descriptions taken in conjunction with the accompanying drawings, which is given by way of illustration only and is not limitative of the present invention.